Compositions and methods regarding engineered and non-engineered gamma delta t-cells for treatment of hematological tumors

Abstract

Aspects of the invention include compositions and methods for treatment of hematological tumors with engineered or non-engineered γδ-T cells. In some embodiments, the γδ-T cells comprise a chimeric antigen receptor (CAR) construct. The CAR construct can contain an anti-CD20 binding domain or anti-B cell maturation antigen (BCMA) binding domain, a CD8 hinge and transmembrane domain, a costimulatory domain, a CD3 ζ signalling domain, a combination thereof, or all thereof. The CAR construct can contain a domain encoding for a secreted common gamma chain cytokine such as a sIL 15 domain.

1 . An isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises
 a. a binding domain that specifically binds to a tumor associated antigen (TAA) expressed on a surface of a hematological tumor cell;   b. a CD8α hinge domain;   c. a CD8α transmembrane domain;   d. a costimulatory signaling region, optionally wherein the costimulatory signaling region is selected from a 4-1BB (CD137) costimulatory signaling region and a CD27 costimulatory signaling region; and   e. a CD3ζ signaling domain.   
     
     
         2 . The isolated nucleic acid sequence of  claim 1 , wherein the (a)-(e) are in 5′ to 3′ order. 
     
     
         3 . The isolated nucleic acid sequence of  claim 1  or  2 , wherein the binding domain specifically binds to CD20. 
     
     
         4 . The isolated nucleic acid sequence of  claim 3 , wherein
 a. the binding domain selectively binds to an epitope within CD20 bound by, or competes for binding with, an anti-CD20 antibody selected from the group consisting of 3B9, 3H7, 2B7, and 9C11, preferably 3H7; and/or   b. the binding domain comprises the complementary determining regions of an anti-CD20 antibody selected from the group consisting of 3B9, 3H7, 2B7, and 9C11, preferably 3H7.   
     
     
         5 . The isolated nucleic acid sequence of any one of  claims 1  to  4 , wherein the binding domain encodes:
 a. a heavy chain variable region (HCVR) sequence and a light chain variable region (LCVR) sequence, wherein the HCVR and LCVR sequences are SEQ ID NO:99 and 107 respectively; 
 b. a heavy chain complementarity determining region 1, 2, and 3 sequence of SEQ ID NOs: 101, 103, and 105 respectively, and a light chain complementarity determining region 1, 2, and 3 sequence of SEQ ID NOs: 109, 111, and 113 respectively; 
 c. a heavy chain complementary determining region 3 (HCDR3) and a light chain CDR3 (LCDR3), wherein the HCDR3 and LCDR3 are selected from the group consisting of SEQ ID NO:345 and 353; 201 and 209; and 249 and 257; 
 d. a heavy chain variable region (HCVR) sequence and a light chain variable region (LCVR) sequence, wherein the HCVR and LCVR sequences are selected from the group consisting of SEQ ID NO: 339 and 347; 195 and 203; and 243 and 251; and/or 
 e. a heavy chain complementary determining region 3 (HCDR3) domain and a light chain CDR3 (LCDR3) domain, wherein the HCDR3 domain comprises an amino acid sequence of the formula X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19, wherein X1=A, V or T; X2=K; X3=D; X4=P, F or G; X5=S or H; X6=Y; X7=G; X8=S or H; X9=G or F; X10=S or Y; X11=Y, N or S; X12=Y, G or H; X13=G, L or S; X14=Y, M or D; X15=Y, D or V; X16=G, V or absent; X17=M or absent; X18=D or absent; X19=V or absent (SEQ ID NO: 369); and the LCDR3 domain comprises an amino acid sequence of the formula X1-X2-X3-X4-X5-X6-X7-X8-X9, wherein X1=Q; X2=Q; X3=R or S; X4=N, Y or F; X5=N, D, or Y; X6=W; X7=P; X8=L; X9=T (SEQ ID NO: 370). 
 
     
     
         6 . The isolated nucleic acid sequence of  claim 1  or  2 , wherein the binding domain specifically binds to CD19 or BCMA. 
     
     
         7 . The isolated nucleic acid sequence of  claim 6 , wherein the binding domain specifically binds to BCMA. 
     
     
         8 . The isolated nucleic acid sequence of  claim 7 , wherein:
 a. the binding domain selectively binds to an epitope within BCMA bound by, or competes for binding with, an anti-BCMA binding region having a sequence selected from the group consisting of SEQ ID NO: 27 and 28; SEQ ID NO: 29 and 30; and SEQ ID NO: and 31 and 32; and/or   b. the binding domain comprises the complementarity determining regions of an anti-BCMA binding region having a sequence selected from the group consisting of SEQ ID NO: 27 and 28; SEQ ID NO: 29 and 30; and SEQ ID NO: and 31 and 32.   
     
     
         9 . The isolated nucleic acid sequence of any one of  claims 1  to  8 , wherein the CAR comprises:
 a. a CD8α hinge domain comprising SEQ ID NO:1 (PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY) or SEQ ID NO:2 (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY); 
 b. a CD8α transmembrane domain comprising SEQ ID NO:3 (IWAPLAGTCGVLLLSLVITLYC); and/or 
 c. a CD3ζ signaling domain comprising: 
 
       
         
           
                 
               
                   (i) 
                 
                   SEQ ID NO: 4 
                 
                   (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQ 
                 
                     
                 
                   RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT 
                 
                     
                 
                   YDALHMQALPPR); 
                 
                   or 
                 
                     
                 
                   (ii) 
                 
                   SEQ ID NO: 5 
                 
                   (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR 
                 
                     
                 
                   RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY 
                 
                     
                 
                   DALHMQALPPR). 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         10 . The isolated nucleic acid sequence of  claim 9 , wherein the CAR comprises:
 a. a 4-1BB costimulatory signaling region comprising SEQ ID NO:6 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL); or   b. a CD27 costimulatory signaling region comprising SEQ ID NO:7. (QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP), or   wherein the isolated nucleic acid encodes the 4-1BB costimulatory signaling region comprising SEQ ID NO:6 and the CD27 costimulatory signaling region comprising SEQ ID NO:7.   
     
     
         11 . The isolated nucleic acid sequence of any one of  claims 1  to  10 , wherein the nucleic acid further encodes:
 a. a secreted cytokine; or 
 b. a secreted common gamma chain interleukin; or 
 c. a secreted IL-15, preferably wherein the IL-15 comprises the sequence of SEQ ID NO:34, more preferably wherein the IL-15 comprises the sequence of SEQ ID NO: 34 operably linked to a secretion signal sequence of SEQ ID NO:33, or wherein the IL-15 comprises the sequence of SEQ ID NO: 34 operably linked to a secretion signal sequence of SEQ ID NO: 49; or 
 d. a secreted common gamma chain interleukin, preferably IL-15, and a multi-cistronic linker region amino terminal to the interleukin or interleukin secretion signal, preferably wherein the multicistronic linker region comprises a sequence of any one of SEQ ID NOs: 43-45, 47, or 52-55 or a combination thereof, or encodes an internal ribosome entry site, e.g., SEQ ID NO: 56 or 60. 
 
     
     
         12 . The isolated nucleic acid sequence of any one of  claims 1  to  5 ,  9  to  10 , or  11 , wherein the binding domain specifically binds to CD20 and the nucleic acid encodes SEQ ID NO:8, 9, 10, 11, 12, 20, 46, 48, or 57 and 58. 
     
     
         13 . The isolated nucleic acid sequence of  claim 12 , wherein the nucleic acid comprises the sequence of SEQ ID NO: 13, 14, 15, 16, 17, 50, 51, or 59. 
     
     
         14 . The isolated nucleic acid sequence of any one of  claims 1  to  2 ,  6  to  10 , or  11 , wherein the binding domain specifically binds to BCMA and the nucleic acid encodes SEQ ID NO: 35, 36, 37, or 38. 
     
     
         15 . The isolated nucleic acid sequence of  claim 14 , wherein the nucleic acid comprises the sequence of SEQ ID NO: 39, 40, 41, or 42. 
     
     
         16 . A polypeptide comprising a chimeric antigen receptor comprising an amino acid sequence encoded by any one of the preceding isolated nucleic acids of  claims 1  to  15 . 
     
     
         17 . A γδ T cell comprising a polypeptide according to  claim 16  or comprising a nucleic acid encoding a CAR construct according to any one of  claims 1  to  15 , wherein the γδ T cell functionally expresses a binding domain of the polypeptide or the nucleic acid encoded CAR on the surface of the γδ T cell. 
     
     
         18 . The γδ T cell of  claim 17 , wherein the γδ T cell exhibits in vitro and/or in vivo cell killing activity against a hematological tumor cell that exhibits cell surface expression of the tumor associated antigen (TAA). 
     
     
         19 . The γδ T cell of  claim 18 , wherein the hematological tumor cell killing activity of said γδ cell is greater than an innate level of in vitro and/or in vivo hematological tumor cell killing activity in a control γδ T cell that does not comprise a CAR construct. 
     
     
         20 . The γδ T cell of  claim 19 , wherein the γδ T cell exhibits the increased hematological tumor cell killing activity against HLA class I +  hematological tumor cells. 
     
     
         21 . The γδ T cell of any one of  claims 18  to  20 , wherein the hematological tumor cell killing activity or increased hematological tumor cell killing activity persists for, for about, for at least, or for at least about, 6 days to 180 days after first contact with the hematological tumor cell. 
     
     
         22 . The γδ T cell of any one of  claims 18  to  21 , wherein the γδ T cell proliferates in response to contact with a hematological tumor cell that exhibits cell surface expression of the tumor associated antigen (TAA). 
     
     
         23 . The γδ T cell of any one of  claims 18  to  22 , wherein the γδ T cell exhibits increased proliferation in response to contact with a hematological tumor cell that exhibits cell surface expression of the tumor associated antigen (TAA) as compared to a control γδ T cell that does not functionally express the nucleic acid encoded CAR on the surface of the γδ T cell. 
     
     
         24 . The γδ T cell of any one of  claims 18  to  23 , wherein the γδ T cell proliferates in a host organism that comprises the hematological tumor cell that exhibits cell surface expression of the tumor associated antigen (TAA). 
     
     
         25 . The γδ T cell of any one of  claims 22  to  24 , wherein the γδ T cell proliferation or increased γδ T cell proliferation persists for, for about, for at least, or for at least about, 6 days to 180 days after first contact with the hematological tumor cell. 
     
     
         26 . The γδ T cell of any one of  claims 17  to  25 , wherein the γδ T cell expresses pro-inflammatory cytokines comprising tumor necrosis factor alpha or interferon gamma after contact with the hematological tumor cell. 
     
     
         27 . The γδ T cell of any one of  claims 17  to  26 , wherein the γδ T cell exhibits reduced, substantially reduced, essentially none, or no graft versus host response when introduced into an allogeneic host in comparison to a graft versus host response exhibited by an αβ T cell administered to an allogeneic host. 
     
     
         28 . The γδ T cell of any one of  claims 17  to  27 , wherein the γδ T cell is a δ1, a δ2, a δ3, or a δ4 γδ T cell, preferably a δ2 −  γδ T cell, more preferably a δ1 γδ T cell. 
     
     
         29 . A plurality of γδ T cells according to any one of  claims 17  to  28 . 
     
     
         30 . The plurality of γδ T cells of  claim 29 , wherein the plurality comprises at least about 10 8  γδ T cells, preferably from about 10 8  γδ T cells to about 10 11  γδ T cells. 
     
     
         31 . The plurality of γδ T cells of  claim 29  or  30 , wherein the plurality comprises a composition that is at least 60%, 80%, or from about 60% or 80% to about 90% or 95% δ1, δ2, δ3, or δ4 γδ T cells, preferably δ1 or δ2 γδ T cells, more preferably δ2 −  γδ T cells, most preferably δ1 γδ T cells. 
     
     
         32 . A method of making the γδ T cell of any one of  claims 17  to  28  or a plurality of γδ T cells of any one of  claims 29  to  31 , wherein the method comprises transfecting γδ T cell (s) with a construct comprising an isolated nucleic acid sequence according to any one of  claims 1  to  15 . 
     
     
         33 . The method of  claim 32 , wherein the method comprises retroviral transduction. 
     
     
         34 . The method of  claim 32  or  33 , wherein the method comprises ex vivo expansion of the γδ T cell(s), wherein the ex vivo expansion is performed before transfection and/or after transfection of the isolated nucleic acid sequence. 
     
     
         35 . A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a γδ T cell of any one of  claims 17  to  28  or a plurality of γδ T cells of any one of  claims 29  to  31 . 
     
     
         36 . A method of killing a hematological tumor cell, the method comprising contacting the hematological tumor cell with a tumor cell killing effective amount of a γδ T cell of any one of  claims 17  to  28 ; a plurality of γδ T cells of any one of  claims 29  to  31 ; or a pharmaceutical composition of  claim 35 . 
     
     
         37 . The method of  claim 36 , wherein the method comprises introducing a therapeutically effective amount of the γδ T cell(s) or the pharmaceutical composition into a host organism comprising the hematological tumor cell. 
     
     
         38 . The method of  claim 37 , wherein the method comprises introducing into a host organism comprising the hematological tumor cell a therapeutically effective amount of the γδ T cell(s) or the pharmaceutical composition and simultaneously or sequentially administering one or more methods to elevate common gamma chain cytokine(s). 
     
     
         39 . The method of  claim 38 , wherein the administering one or more methods to elevate common gamma chain cytokine(s) comprises administering simultaneously with introducing the γδ T cell (s) or sequentially an amount of common gamma chain cytokine(s) effective to increase proliferation, cytotoxic activity, persistence, or the combination thereof of the introduced γδ T cell (s), preferably wherein the method comprises administering IL-2, more preferably wherein the method comprises administering IL-15. 
     
     
         40 . The method of  claim 39 , wherein the one or more methods to elevate common gamma chain cytokine(s) comprise administering an amount of common gamma chain cytokine(s) effective to increase proliferation, cytotoxic activity, persistence, or the combination thereof of the introduced γδ T cell(s) before and/or after introducing the γδ T cell (s). 
     
     
         41 . The method of any one of  claims 38  to  40 , wherein the one or more methods to elevate common gamma chain cytokine(s) comprises lymphodepletion before introducing the γδ T cell(s). 
     
     
         42 . The method of any one of  claims 38  to  40 , wherein the one or more methods to elevate common gamma chain cytokine(s) comprises secretion of one or more common gamma chain cytokine(s) from the introduced γδ T cell (s). 
     
     
         43 . The method of any one of  claims 37  to  42 , wherein the method reduces the in vivo tumor burden in the host organism, and/or increases the mean survival time of the host organism as compared to a control organism, wherein the control organism is not treated with the γδ T cell(s) or the pharmaceutical composition. 
     
     
         44 . The method of any one of  claims 36  to  43 , wherein the method is a method of treating cancer in a subject in need thereof. 
     
     
         45 . Use of a tumor cell killing effective amount of a γδ T cell of any one of  claims 17  to  28 ; a plurality of γδ T cells of any one of  claims 29  to  31 ; or a pharmaceutical composition of  claim 35  in the manufacture of a medicament for the treatment of a hematological tumor cell cancer in a subject in need thereof. 
     
     
         46 . A method of treating cancer in a subject in need thereof, the method comprising:
 a. administering a therapeutically effective amount of γδ T cells, wherein the cancer comprises hematological tumor cells that exhibit cell surface expression of CD20; or   b. administering a therapeutically effective amount of γδ T cells, wherein the cancer comprises hematological tumor cells that exhibit cell surface expression of BCMA.   
     
     
         47 . The method of  claim 46 , wherein the method comprises simultaneously with the administering of γδ T cells or sequentially, administering one or more methods to elevate common gamma chain cytokine(s). 
     
     
         48 . The method of  claim 46  or  47 , wherein the method comprises performing a plurality of administrations of the γδ T cells, wherein the interval between the plurality of administrations is at least about a week, preferably at least about 2, 3, 4, 5, 6, 7, 8, or 12 weeks, and/or no more than once every 6 or 12 months. 
     
     
         49 . A pharmaceutical composition for use in any one of the methods of  claims 46  to  48 .